Ordered insertional mutagenesis at a single genomic site enables lineage tracing and analog recording in mammalian cells

The study of intricate cellular and developmental processes in the context of complex multicellular organisms is difficult because it may require the non-destructive observation of thousands, millions, or even billions of cells deep within an animal. To overcome this difficulty, several groups have recently reported CRISPR-based DNA recorders that convert transient cellular experiences or processes into durable genomic mutations, which can then be read by next-generation sequencing in high-throughput. However, existing DNA recorders rely primarily on the accumulation of CRISPR-induced deletion mutations, which can be problematic because in the limit of progressive deletion mutations, no record remains. Here, we present a high-information DNA recorder that accumulates insertion mutations in temporal order at a single locus. Our recorder, called CHYRON (Cell HistorY Recording by Ordered iNsertion), can be applied as an evolving lineage tracer as well as a cellular stimulus recorder. As a lineage tracer, CHYRON allowed us to perfectly reconstruct the lineage relationships among 16 populations of human cells descended from four starting populations that were subject to a series of splitting steps. In this experiment, CHYRON progressively accumulated and retained insertions in 20% percent of cells such that the average length of insertion generated was 8.4 bp (~15 bits), reflecting high information content. As a stimulus recorder, we show that when the CHYRON machinery is placed under the control of a stress-responsive promoter, the frequency and lengths of insertions reflect the dose and duration of the stress. With further engineering of CHYRON9s components to increase encoding capabilities and reduce loss, CHYRON9s special ability to progressively accumulate insertion mutations should lead to single-cell-resolution recording of lineage and other information through long periods of time in complex animals or tumors, ultimately providing a full picture of mammalian development.